ABSTRACT Cystic fibrosis (CF) is a life-shortening inherited disease caused by the loss or dysfunction of the CF transmembrane conductance regulator (CFTR) channel activity resulting from mutations. Clinically, chronic lung disease is the main cause of morbidity and mortality for CF patients. Among the 2000+ disease-causing mutations, ?F508 is the most common mutation and associates with a severe form of CF disease. The ideal therapy for CF associated with ?F508 requires increasing the quantity of ?F508-CFTR protein at the plasma membrane, potentiating the impaired channel gating properties, and improving its stability. This notion was supported by the approval of two CFTR modulating drugs, Orkambi (VX-809 + VX-770) and Symdeko (VX- 661 + VX-770), to treat CF patients homozygous for ?F508, and by trials with triple combinations (VX-445 + VX- 661 + VX-770; VX-659 + VX-661 + VX-770). It is to be noted that the clinical benefits of approved drugs are modest and the mechanisms of action of these CFTR correctors are poorly understood. In this grant, we plan to study the mechanisms of how CFTR correctors promote the maturation of ?F508-CFTR and stabilize the mutant protein at the plasma membrane. We will focus on VX- CFTR correctors (VX-661, VX-809, VX-445, and VX-659) and study the subject from the perspective of CFTR-containing macromolecular complexes. The hypotheses to be tested are: (i) CFTR correctors (e.g., VX- CFTR correctors) bind directly to ?F508-CFTR to exert their rescue effects. A high-affinity binding will produce a better rescue outcome. (ii) The instability of ?F508-CFTR (with a short half-life) at the plasma membrane is, at least in part, due to its reduced ability to interact with binding partners and consequently cannot form a stable macromolecular complex, which leads to its rapid internalization and targeted for degradation. (iii) CFTR correctors not only help fold ?F508-CFTR in the ER to promote its maturation, but also stabilize the mutant protein at the plasma membrane by enhancing its interaction with binding partners and facilitating the formation of a stable macromolecular complex. And (iv) by isolating the corrector-associated- and ?F508-CFTR-containing complexes under different conditions and using proteomics, we can identify effectors and pathways important in the rescuing process. Click chemistry and photo-affinity labeling will be used to investigate the interactions and macromolecular complexes formation in various CF model systems. This study will help us (i) better understand the mechanisms of action of CFTR correctors, (ii) identify novel targets in ?F508-CFTR-containing complexes, (iii) develop more potent drugs to combat CF, and (iv) understand the molecular basis of other human diseases resulting from insufficiently folded and processed proteins (e.g., P-glycoprotein) and find ways to treat these diseases.